The vitamin D receptor (VDR) is a transcription factor that mediates the actions of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in the nucleus of target cells. The receptor binds in a cell-specific manner to target genes and regulates their transcriptional output. The VDR gene is expressed in numerous cell types, and its concentration is regulated by multiple factors. Our recent studies have defined several critical regions within the VDR gene that are crucial to the expression and regulation of the VDR. The importance of the VDR as a determinant of response to the vitamin D hormone dictates three specific aims. Aim 1: To assess the autonomous capabilities of mouse and human VDR gene loci to direct tissue-specific and regulatory expression of the VDR gene in vivo. BAC clone transgenesis will be conducted to prepare mouse strains containing integrated copies of both mouse and human VDR gene loci. The capacity of these transgenes to direct tissue- specific expression of VDR activity will be evaluated through both reporter gene analysis (tissue luciferase activity) and VDR protein production. Cellular expression will be assessed using immunohistological evaluation of both reporter gene and epitope-tagged VDR expression. The capacity of these transgenes to modulate VDR gene expression in response to hormonal regulators will also be examined. Aim 2: To define the VDR gene- specific regulome and to characterize key determinants of both tissue-specific expression and regulatory control of the VDR gene. We propose to utilize both chromatin immunoprecipitation (ChIP)/high density DNA microarray (chip) (ChIP-chip) analyses in both cells in culture and in mice in vivo together with bacterial artificial chromosome (BAC) analysis in cells in vitro to identify the key regulatory regions and the transacting factors that are involved in both tissue-specific VDR expression and regulation in bone, kidney and intestine. Aim 3: To link individual VDR gene enhancers with properties of tissue-specific and 1,25(OH)2D3 regulatory control in vivo. The ability of individual enhancers to direct tissue-specific expression of the VDR will be assessed in vivo through VDR gene BAC clone transgenesis. VDR gene enhancers or regions with potentially unique tissue-specific properties as identified in Aim 2 will be individually deleted from the wildtype mouse VDR BAC clone and their reporter and VDR protein expression activities assessed following preparation of corresponding transgenic mouse strains. We will also explore the biologic consequence of VDR gene autoregulation by 1,25(OH)2D3 in vivo. VDR gene expression is essential to vitamin D response;its upregulation enhances the hormone's efficacy in controlling numerous biological processes. These novel studies will provide new insight into mechanisms that govern VDR gene's transcriptional activity. PUBLIC HEALTH RELEVANCE: Vitamin D plays significant roles through its receptor both in the maintenance of mineral homeostasis and in the control of cellular growth and function. The studies herein seek to determine the mechanisms that control expression of the vitamin D receptor in tissues such that better and more selective medicines can be created.